Chorismate biosynthesis enzymes

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a chorismate synthase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the chorismate synthase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the chorismate synthase in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/093,611, filed Jul. 21, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingenzymes involved in chorismate biosynthesis in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Chorismate biosynthesis involves the last few steps in the commonpathway for the production of the aromatic amino acids phenylalanine,tyrosine and tryptophan. The last common step of biosynthesis ofaromatic amino acids produced via the shikimic acid pathway is catalyzedby chorismate synthase which produces chorismate from5-enolpyruvylshikimate 3-phosphate. The enzyme requires reduced FMN as acofactor. There are two forms of the enzyme described in tomato. Tomatochorismate synthase 1 and 2 have 88% identity at the amino acid leveland are expressed differentially in flowers, roots and stems (Gorlach,J. and Schmid, J. (1993) Plant Mol Biol 23:707-716).

[0004] Manipulating either the amount or activity of this enzyme wouldafford manipulation of the ratio of aromatic to non-aromatic amino acidsin plants, including corn, rice, soybean and wheat. This enzyme shouldalso be useful for high throughput screening of compounds suitable foruse as herbicides.

SUMMARY OF THE INVENTION

[0005] The instant invention relates to isolated nucleic acid fragmentsencoding chorismate synthase. Specifically, this invention concerns anisolated nucleic acid fragment encoding a chorismate synthase and anisolated nucleic acid fragment that is substantially similar to anisolated nucleic acid fragment encoding a chorismate synthase. Inaddition, this invention relates to a nucleic acid fragment that iscomplementary to the nucleic acid fragment encoding chorismate synthase.

[0006] An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a chorismatesynthase.

[0007] In another embodiment, the instant invention relates to achimeric gene encoding a chorismate synthase, or to a chimeric gene thatcomprises a nucleic acid fragment that is complementary to a nucleicacid fragment encoding a chorismate synthase, operably linked tosuitable regulatory sequences, wherein expression of the chimeric generesults in production of levels of the encoded protein in a transformedhost cell that is altered (i.e., increased or decreased) from the levelproduced in an untransformed host cell.

[0008] In a further embodiment, the instant invention concerns atransformed host cell comprising in its genome a chimeric gene encodinga chorismate synthase, operably linked to suitable regulatory sequences.Expression of the chimeric gene results in production of altered levelsof the encoded protein in the transformed host cell. The transformedhost cell can be of eukaryotic or prokaryotic origin, and include cellsderived from higher plants and microorganisms. The invention alsoincludes transformed plants that arise from transformed host cells ofhigher plants, and seeds derived from such transformed plants.

[0009] An additional embodiment of the instant invention concerns amethod of altering the level of expression of a chorismate synthase in atransformed host cell comprising: a) transforming a host cell with achimeric gene comprising a nucleic acid fragment encoding a chorismatesynthase; and b) growing the transformed host cell under conditions thatare suitable for expression of the chimeric gene wherein expression ofthe chimeric gene results in production of altered levels of chorismatesynthase in the transformed host cell.

[0010] An addition embodiment of the instant invention concerns a methodfor obtaining a nucleic acid fragment encoding all or a substantialportion of an amino acid sequence encoding a chorismate synthase.

[0011] A further embodiment of the instant invention is a method forevaluating at least one compound for its ability to inhibit the activityof a chorismate synthase, the method comprising the steps of: (a)transforming a host cell with a chimeric gene comprising a nucleic acidfragment encoding a chorismate synthase, operably linked to suitableregulatory sequences; (b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of chorismatesynthase in the transformed host cell; (c) optionally purifying thechorismate synthase expressed by the transformed host cell; (d) treatingthe chorismate synthase with a compound to be tested; and (e) comparingthe activity of the chorismate synthase that has been treated with atest compound to the activity of an untreated chorismate synthase,thereby selecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS

[0012] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0013] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. § 1.821-1.825. TABLE 1 Chorismate Synthase SEQ ID NO:Plant Clone Designation (Nucleotide) (Amino Acid) Cornchpc24.pk0002.h1:fis 1 2 Soybean sl1.pk0143.g5:fis 3 4 Wheatwre1n.pk0094.e6 5 6 Corn csi1n.pk0050.d11:fis 7 8 Rice rls48.pk0033.g1 910 Rice rls72.pk0029.g8 11 12 Soybean ses9c.pk001.j6 13 14

[0014] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. § 1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0015] In the context of this disclosure, a number of terms shall beutilized. As used herein, a “nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A nucleic acidfragment in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

[0016] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-á-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof.

[0017] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by nucleic acid fragments that do not share 100%sequence identity with the gene to be suppressed. Moreover, alterationsin a nucleic acid fragment which result in the production of achemically equivalent amino acid at a given site, but do not effect thefunctional properties of the encoded polypeptide, are well known in theart. Thus, a codon for the amino acid alanine, a hydrophobic amino acid,may be substituted by a codon encoding another less hydrophobic residue,such as glycine, or a more hydrophobic residue, such as valine, leucine,or isoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts.

[0018] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0019] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0020] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0021] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0022] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0023] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0024] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0025] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic nucleotide segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a nucleic acid fragmentto be expressed in most cell types at most times are commonly referredto as “constitutive promoters”. New promoters of various types useful inplant cells are constantly being discovered; numerous examples may befound in the compilation by Okamuro and Goldberg (1989) Biochemistry ofPlants 15:1-82. It is further recognized that since in most cases theexact boundaries of regulatory sequences have not been completelydefined, nucleic acid fragments of different lengths may have identicalpromoter activity.

[0026] The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

[0027] The “3′ non-coding sequences” refer to nucleotide sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0028] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0029] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single nucleic acid fragment so thatthe function of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0030] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0031] “Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

[0032] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of MRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0033] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0034] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

[0035] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0036] Nucleic acid fragments encoding at least a portion of severalchorismate synthases have been isolated and identified by comparison ofrandom plant cDNA sequences to public databases containing nucleotideand protein sequences using the BLAST algorithms well known to thoseskilled in the art. The nucleic acid fragments of the instant inventionmay be used to isolate cDNAs and genes encoding homologous proteins fromthe same or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

[0037] For example, genes encoding other chorismate synthases, either ascDNAs or genomic DNAs, could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

[0038] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theinstant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673; Loh et al. (1989)Science 243:217). Products generated by the 3′ and 5′ RACE procedurescan be combined to generate full-length cDNAs (Frohman and Martin (1989)Techniques 1:165).

[0039] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36: 1; Maniatis).

[0040] The nucleic acid fragments of the instant invention may be usedto create transgenic plants in which the disclosed polypeptides arepresent at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the ratio of aromatic to non-aromatic aminoacids in those cells. This may also create plants that are resistant toherbicides.

[0041] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

[0042] Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

[0043] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

[0044] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0045] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0046] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppresiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds, and is notan inherent part of the invention. For example, one can screen bylooking for changes in gene expression by using antibodies specific forthe protein encoded by the gene being suppressed, or one could establishassays that specifically measure enzyme activity. A preferred methodwill be one which allows large numbers of samples to be processedrapidly, since it will be expected that a large number of transformantswill be negative for the desired phenotype.

[0047] The instant polypeptide (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptide of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptide are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptide.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded chorismate synthase. An example of a vector for high levelexpression of the instant polypeptide in a bacterial host is provided(Example 6).

[0048] Additionally, the instant polypeptide can be used as a target tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptide described herein catalyzes the formation of the last commonprecursor precursor in the biosynthesis of numerous aromatic compounds.

[0049] Accordingly, inhibition of the activity of the enzyme describedherein could lead to inhibition plant growth. Thus, the instantpolypeptide could be appropriate for new herbicide discovery and design.

[0050] All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J Hum. Genet.32:314-331).

[0051] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bematzky and Tanksley (1986) Plant Mol.Biol. Reporter 4(1):37-41. Numerous publications describe geneticmapping of specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0052] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0053] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:14-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Research5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

[0054] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 17:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0055] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Nati. Acad. Sci USA 86:9402; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptide disclosed herein.

EXAMPLES

[0056] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions.

Example 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

[0057] cDNA libraries representing mRNAs from various corn, rice,soybean and wheat tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 cDNA Libraries from Corn, Rice,Soybean and Wheat Library Tissue Clone chpc24 Corn (MBS847) 8 Day OldShoot Treated Chpc24.pk0002.h1 24 Hours With Herbicide* csi1n CornSilk** csi1n.pk0050.d11 rls48 Rice Leaf 15 Days After Germination, 48rls48.pk0033.g1 Hours After Infection of Strain Magaporthe grisea4360-R-67 (AVR2- YAMO); Susceptible rls72 Rice Leaf 15 Days AfterGermination, 72 rls72.pk0029.g8 Hours After Infection of StrainMagaporthe grisea 4360-R-67 (AVR2- YAMO); Susceptible ses9c SoybeanEmbryogenic Suspension ses9c.pk001.j6 sl1 Soybean Two-Week-OldDeveloping sl1.pk0143.g5 Seedlings wre1n Wheat Root From 7 Day OldEtiolated wre1n.pk0094.e6 Seedling**

[0058] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAPT™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651). The resulting ESTs are analyzed using a Perkin Elmer Model377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0059] cDNA clones encoding chorismate synthase were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) NatureGenetics 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Chorismate Synthases

[0060] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs tochorismate synthases from Lycopersicon esculentum (NCBI GeneralIdentifier Nos. 2492952 and 2492953). Shown in Table 3 are the BLASTresults for individual ESTs (“EST”), or the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”): TABLE 3 BLASTResults for Sequences Encoding Polypeptides Homologous to ChorismateSynthases NCBI General BLAST Clone Status Identifier No. pLog Scorechpc24.pk0002.h1 FIS 2492952 254.00 sl1.pk0143.g5 FIS 2492952 43.52wre1n.pk0094.e6 FIS 2492952 104.00 csi1n.pk0050.d11 FIS 2492953 172.00rls48.pk0033.g1 EST 2492953 26.15 rls72.pk0029.g8 EST 2492953 127.00ses9c.pk001.j6 EST 2492953 62.10

[0061] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode two entire corn chorismate synthases whichcorrespond to the two known tomato chorismate synthase isozymes (LeCS 1and LeCS2); a substantial portion of two soybean chorismate synthases;two portions of rice chorismate synthase (may be the same or differentgenes); and a substantial portion of a wheat chorismate synthase. Thesesequences represent the first corn, rice, soybean and wheat sequencesencoding chorismate synthase.

Example 4 Expression of Chimeric Genes in Monocot Cells

[0062] A chimeric gene comprising a cDNA encoding the instantpolypeptide in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and Smal and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase T DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptide, and the 10 kD zein 3′ region.

[0063] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0064] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35 S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0065] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0066] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0067] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0068] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

[0069] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptide in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

[0070] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0071] Soybean embroys may then be transformed with the expressionvector comprising sequences encoding the instant polypeptide. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0072] Soybean embryogenic suspension cultures can maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0073] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0074] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0075] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0076] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0077] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

[0078] The cDNAs encoding the instant polypeptide can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0079] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0080] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL2 1 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 1L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

Example 7 Evaluating Compounds for Their Ability to Inhibit the Activityof Chorismate Synthase

[0081] The polypeptide described herein may be produced using any numberof methods known to those skilled in the art. Such methods include, butare not limited to, expression in bacteria as described in Example 6, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptide may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yieid intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

[0082] Purification of the instant polypeptide, if desired, may utilizeany number of separation technologies familiar to those skilled in theart of protein purification. Examples of such methods include, but arenot limited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptide are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptide may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose4B. In an alternate embodiment, athioredoxin fusion protein may be eluted using dithiothreitol; however,elution may be accomplished using other reagents which interact todisplace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

[0083] Crude, partially purified or purified enzyme, either alone or asa fusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptide disclosed herein. Assays may be conducted under wellknown experimental conditions which permit optimal enzymatic activity.Assays for chorismate synthase are presented by F. Gibson (1970) MethodsEnzymol. 17:362-364, White, P. J., Mousdale, D. M. and Coggins, J. R.(1987) Biochem. Soc. Trans. 15:144-145, Schaller, A., Windhofer, V. andAmrhein, N. (1990) Arch. Biochem. Biophys. 282:437442 and Boocock, M. R.and Coggins, J. R. (1983) FEBS Lett. 154:127-133.

1 14 1 1635 DNA Zea mays 1 gcacgagcgc agtctcagac cctcaccaac caggcaaccaaaccttctcc gatggccgcg 60 cccgtgtcgc agccgccggt gtccgccagg gcgtccacacggtttctccc ccgcgggata 120 ggcgcgctcc cggagtccgc ccccacgtcc ctccggttatccgtcggccg ccgtcgccgc 180 gcctccagcc tagaggtgaa ggcatcagga aatgtgttcgggaactactt ccaggttgca 240 acctatggcg aatcccatgg agggggtgtt ggttgcgttatcagtggctg cccacccaga 300 attcctctca ctgaggcaga catgcaagta gaactcgatagaagacgtcc gggtcaaagt 360 agaattacaa ccccaagaaa ggagactgat acatgcaaaattctatcagg gacacatgat 420 gggatgacta ctggtacacc aattcacgtc tttgtcccaaacacagatca aaggggtggt 480 gattacagtg aaatgtctaa ggcgtacaga ccatcccatgcagatgcaac ctatgacttc 540 aagtatggag ttagagctgt gcagggaggt ggaaggtcatcagccagaga aaccattggc 600 agggtggctg caggagctct tgcaaagaaa attctaaagctcaaatcagg agtggagatc 660 ttggcatttg tttctaaagt gcaccaagtc gtacttccagaagatgcagt tgattatgag 720 actgtaacct tggaacatat agagagcaac atcgttagatgtcctgatcc agaatatgca 780 gagaagatga ttgctgccat tgatacggta cgagttagaggagattcaat tggtggggtc 840 gtcacatgca ttgcaagaaa tgttcctcgt ggtcttggctctcctgtttt tgacaaactt 900 gaagctgaac tggcaaaagc catgctttct cttcctgcaagcaaggggtt tgagattggc 960 agtgggttcg ctggtacgga ctttactgga agtgagcataatgatgagtt ctatatggat 1020 gaggctggaa atgtgaggac acgaactaat cgctcaggcggtgttcaggg agggatatca 1080 aatggtgaaa ttatttactt caaagtggct tttaagccaacagcaactat cggaaagaag 1140 caaaatactg tgtcaaggga gcatgaggat gttgaacttttggcaagggg gcgccatgac 1200 ccctgtgttg tccctcgagc tgttcctatg gtggaatccatggctgcgct ggtcctgatg 1260 gaccagctca tggcgcatat tgcccagtgt gagatgtttccgctgaacct tgccctacaa 1320 gagcccattg gctctgctag cagtgcatct gaactgtcaccaaacctatc ataatgtttg 1380 tcgtggaaca tgtcccagct ttccttctat cgaaattctggtctttgcta agcagtttgc 1440 aattcggaac ccccataaac cctcgactat tgtacctagagataaagtga acggatatca 1500 agatagaaat gcattaatgt ttttgtgatg tgtagtataactgatattta ccccttttct 1560 ttttttgaga gaggacgcat gatgtcgttt gagcaataaagtttaatttg ggagaaaaaa 1620 aaaaaaaaaa aaaaa 1635 2 440 PRT Zea mays 2Met Ala Ala Pro Val Ser Gln Pro Pro Val Ser Ala Arg Ala Ser Thr 1 5 1015 Arg Phe Leu Pro Arg Gly Ile Gly Ala Leu Pro Glu Ser Ala Pro Thr 20 2530 Ser Leu Arg Leu Ser Val Gly Arg Arg Arg Arg Ala Ser Ser Leu Glu 35 4045 Val Lys Ala Ser Gly Asn Val Phe Gly Asn Tyr Phe Gln Val Ala Thr 50 5560 Tyr Gly Glu Ser His Gly Gly Gly Val Gly Cys Val Ile Ser Gly Cys 65 7075 80 Pro Pro Arg Ile Pro Leu Thr Glu Ala Asp Met Gln Val Glu Leu Asp 8590 95 Arg Arg Arg Pro Gly Gln Ser Arg Ile Thr Thr Pro Arg Lys Glu Thr100 105 110 Asp Thr Cys Lys Ile Leu Ser Gly Thr His Asp Gly Met Thr ThrGly 115 120 125 Thr Pro Ile His Val Phe Val Pro Asn Thr Asp Gln Arg GlyGly Asp 130 135 140 Tyr Ser Glu Met Ser Lys Ala Tyr Arg Pro Ser His AlaAsp Ala Thr 145 150 155 160 Tyr Asp Phe Lys Tyr Gly Val Arg Ala Val GlnGly Gly Gly Arg Ser 165 170 175 Ser Ala Arg Glu Thr Ile Gly Arg Val AlaAla Gly Ala Leu Ala Lys 180 185 190 Lys Ile Leu Lys Leu Lys Ser Gly ValGlu Ile Leu Ala Phe Val Ser 195 200 205 Lys Val His Gln Val Val Leu ProGlu Asp Ala Val Asp Tyr Glu Thr 210 215 220 Val Thr Leu Glu His Ile GluSer Asn Ile Val Arg Cys Pro Asp Pro 225 230 235 240 Glu Tyr Ala Glu LysMet Ile Ala Ala Ile Asp Thr Val Arg Val Arg 245 250 255 Gly Asp Ser IleGly Gly Val Val Thr Cys Ile Ala Arg Asn Val Pro 260 265 270 Arg Gly LeuGly Ser Pro Val Phe Asp Lys Leu Glu Ala Glu Leu Ala 275 280 285 Lys AlaMet Leu Ser Leu Pro Ala Ser Lys Gly Phe Glu Ile Gly Ser 290 295 300 GlyPhe Ala Gly Thr Asp Phe Thr Gly Ser Glu His Asn Asp Glu Phe 305 310 315320 Tyr Met Asp Glu Ala Gly Asn Val Arg Thr Arg Thr Asn Arg Ser Gly 325330 335 Gly Val Gln Gly Gly Ile Ser Asn Gly Glu Ile Ile Tyr Phe Lys Val340 345 350 Ala Phe Lys Pro Thr Ala Thr Ile Gly Lys Lys Gln Asn Thr ValSer 355 360 365 Arg Glu His Glu Asp Val Glu Leu Leu Ala Arg Gly Arg HisAsp Pro 370 375 380 Cys Val Val Pro Arg Ala Val Pro Met Val Glu Ser MetAla Ala Leu 385 390 395 400 Val Leu Met Asp Gln Leu Met Ala His Ile AlaGln Cys Glu Met Phe 405 410 415 Pro Leu Asn Leu Ala Leu Gln Glu Pro IleGly Ser Ala Ser Ser Ala 420 425 430 Ser Glu Leu Ser Pro Asn Leu Ser 435440 3 763 DNA Glycine max 3 gaacaagaac aaatcgctct ggtgggatac agggtggaatttccaatggg gaaatcatta 60 atatgagaat agctttcaag ccaacatcaa caattggaaagaagcaaaag actgtgactc 120 gagataaaaa agaaacagag tttatagccc gtggtcgccatgatccttgt gttgtcccaa 180 gagctgtacc tatggtagaa gcaatggtag ctttggttcttgtggaccaa ttgatggcac 240 aatatgcgca gtgtaatctt tttcccgtaa actcagatttgcaagaaccc ttggtgccca 300 tactacggcc agaagaagcg ctcctctgaa gaggaagggggtccataaat cagtaattgg 360 cctttgataa aatcttcctt atggctagtg tttaattgacacggttaatt cactttgatg 420 acaagtccaa gtgaacattg tggcagatat ttttgcgggtgcaatctatc gttttgtatt 480 aatgtaagtt aaactatgtt ttcttttcct ctcttcttctattttcattc tgagggtgaa 540 cattgtttct agtaaacctt gttgcaaaag cagagatagatgtattctta aagtgaactg 600 atattaaaaa ttgtaagaaa cgtatcagtt tttgggcttaataagtgttg ctctgctttg 660 caataaatga aagctttggc aactttaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 720 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaat 763 4 108 PRT Glycine max 4 Thr Arg Thr Asn Arg Ser Gly Gly Ile GlnGly Gly Ile Ser Asn Gly 1 5 10 15 Glu Ile Ile Asn Met Arg Ile Ala PheLys Pro Thr Ser Thr Ile Gly 20 25 30 Lys Lys Gln Lys Thr Val Thr Arg AspLys Lys Glu Thr Glu Phe Ile 35 40 45 Ala Arg Gly Arg His Asp Pro Cys ValVal Pro Arg Ala Val Pro Met 50 55 60 Val Glu Ala Met Val Ala Leu Val LeuVal Asp Gln Leu Met Ala Gln 65 70 75 80 Tyr Ala Gln Cys Asn Leu Phe ProVal Asn Ser Asp Leu Gln Glu Pro 85 90 95 Leu Val Pro Ile Leu Arg Pro GluGlu Ala Leu Leu 100 105 5 1015 DNA Triticum aestivum 5 gcacgaggctgcaggagctg ttgcaaagaa aattcttaag ctgaaatgtg gagtagagat 60 tctagcatttgtttccaaag tgcatcaagt ggtacttcct gaagacgcag ttgattatga 120 aactcttaccctggatcaga tagagagcaa catttgtaga tgtcctgatc cagaatatgc 180 acagaagatgattgatgcaa ttgataaagt acgagttaat gggaattcga ttggtggggt 240 ggtcacatgcattgccagaa atgttcctcg tgggcttggc tctcctgtat ttgacaaact 300 tgaagctctactggcaaagg ctatgctttc tcttcctgca agcaaggggt ttgagatcgg 360 tagtggatttgcaggtactg acctaactgg aagtgagcat aacgatgagt tctatatgga 420 cgaggctggaaatgtaagaa cacgaaccaa tcgctcgggc ggtgtacagg gagggatatc 480 aaatggtgaaactatatact tcaaagtagc tttcaagcca acagcaacta ttgggaagaa 540 gcaaaatactgtaacaaggg atcatgagga tatcgaactt ctgacaaggg gtcgccatga 600 cccatgtgtcgtccctcggg ctgttccaat ggtggagacg atggctgcat tggtcctcat 660 ggaccagctgatggcacatg ttgctcagtg cgagatgttc ccgctgaacc tcgccctaca 720 agaaccaatcggctccgcaa acagtacacc tgcgttggca ccagatctag catgatgccc 780 gccttggaacgagaaggccc tcttacatat tcttctccct ctctttcatc tgccacattt 840 cagggttttgctaagcagtt tgcagttttg taccaaacct gtatcctaga atatacattg 900 gattagtgtacaccaagaga tctgttgatc accgaaaata aaagtttgcg gcgtgaacag 960 tttgttctggacaaccagtc aatgtgagca atagaacatt tctccacttg ttgaa 1015 6 257 PRTTriticum aestivum 6 His Glu Ala Ala Gly Ala Val Ala Lys Lys Ile Leu LysLeu Lys Cys 1 5 10 15 Gly Val Glu Ile Leu Ala Phe Val Ser Lys Val HisGln Val Val Leu 20 25 30 Pro Glu Asp Ala Val Asp Tyr Glu Thr Leu Thr LeuAsp Gln Ile Glu 35 40 45 Ser Asn Ile Cys Arg Cys Pro Asp Pro Glu Tyr AlaGln Lys Met Ile 50 55 60 Asp Ala Ile Asp Lys Val Arg Val Asn Gly Asn SerIle Gly Gly Val 65 70 75 80 Val Thr Cys Ile Ala Arg Asn Val Pro Arg GlyLeu Gly Ser Pro Val 85 90 95 Phe Asp Lys Leu Glu Ala Leu Leu Ala Lys AlaMet Leu Ser Leu Pro 100 105 110 Ala Ser Lys Gly Phe Glu Ile Gly Ser GlyPhe Ala Gly Thr Asp Leu 115 120 125 Thr Gly Ser Glu His Asn Asp Glu PheTyr Met Asp Glu Ala Gly Asn 130 135 140 Val Arg Thr Arg Thr Asn Arg SerGly Gly Val Gln Gly Gly Ile Ser 145 150 155 160 Asn Gly Glu Thr Ile TyrPhe Lys Val Ala Phe Lys Pro Thr Ala Thr 165 170 175 Ile Gly Lys Lys GlnAsn Thr Val Thr Arg Asp His Glu Asp Ile Glu 180 185 190 Leu Leu Thr ArgGly Arg His Asp Pro Cys Val Val Pro Arg Ala Val 195 200 205 Pro Met ValGlu Thr Met Ala Ala Leu Val Leu Met Asp Gln Leu Met 210 215 220 Ala HisVal Ala Gln Cys Glu Met Phe Pro Leu Asn Leu Ala Leu Gln 225 230 235 240Glu Pro Ile Gly Ser Ala Asn Ser Thr Pro Ala Leu Ala Pro Asp Leu 245 250255 Ala 7 1626 DNA Zea mays 7 gcacgagctc agcttcgtct ctctcgccggcgcggcaggc aactatcatc acttcattag 60 ctcatccaat ctattccgat gacgaccgtgcccaagccac agcaggtggc gcactcacgg 120 gcacggctcg caccccgcgc gatcggcgccttgctggagt ttgccccagc ctcctcctcc 180 ctccgcttcg ccgtgcaccg ctgccgcactgctcgcctag aggtgaaggc atctggaaac 240 acgtttggaa actactttca ggttgcaacctatggtgaat ctcatggggg tggtgttggt 300 tgtgttatca gtggttgtcc acctagaattccactcactg aggcagacct acaagttgaa 360 ctcgatcgaa gacggcccgg acagagcagaataacctcca caaggaagga gactgataca 420 tgcaaaattc tgtcagggac acatgaaggggtgactactg gaacgccaat tcttgttatt 480 gtcccaaaca cagatcaaat aggcagtgatcaccgtgaaa tagccaatgt gtaccgacct 540 tctcatgcag acgcaactta tgacttcaagtacggtgtta gagctgtaca gggaggtggg 600 aggtcctcgg gcagaaaaac cgttggaagggtggctgcag gggccctccc caagaaaatt 660 cttaagctca aatgtggatt agagatcttgtcgtttgttt ccaaagtgca tcaggttgtg 720 ctcccagaag acgcggttga ttatgggtctgtaactttgg aacagataga gagcaacatc 780 gttagatgtc ctgatccaga gtacgcagagaagatgatag acgcaatcga cagagtacga 840 gttcgagggg attcggtcgg tggagtgatcacatgcgtcg ctagaaacgt tcctcgcggg 900 ctcggttctc ctgtgttcga caagctcgaatccgaactgg caaaagctat gctttctatt 960 cctgcgagca acgggttcga gattggcagcggattcgccg ggaccgactt gacaggaagt 1020 gagcataatg atgagtttta tatggataaggctggaagtg tcaggacacg gactaatcgc 1080 tcgggtggtg tgcagggagg gatatcgaatgttgagattg tgcacttcaa agttgctttt 1140 aagccgacac catctatcgg ggtgaaacagaacaccgtgt caagggagcg tcagaacgtt 1200 gagcttctag caagagggcg ccatgacccatgcgtcgccc ctcgagctgt tcctgtggtg 1260 gaatccatgg ccgcgttggt cctcatggaccagctgatgg cgcacgtggc tcagtgcgag 1320 atgttcgcgc tcaatactgc acttcaagaaccagttggct ctttctagca gaggcagagc 1380 acacctgatg agctcgcgcc aaattttatcatttatcata gtaataagta gctcaagcgt 1440 ggcttggttt gcttgtctct tgcaccgtagttttgttttt tttttcccgc aagtgtgatg 1500 cgatgaagtg aataaggcac ttggtttcctgtgcatttgt acacgtttca tataatgtaa 1560 tctacttcga agatgatgca tttttatagatgtggcttgt gaaagacaaa aaaaaaaaaa 1620 aaaaaa 1626 8 429 PRT Zea mays 8Met Thr Thr Val Pro Lys Pro Gln Gln Val Ala His Ser Arg Ala Arg 1 5 1015 Leu Ala Pro Arg Ala Ile Gly Ala Leu Leu Glu Phe Ala Pro Ala Ser 20 2530 Ser Ser Leu Arg Phe Ala Val His Arg Cys Arg Thr Ala Arg Leu Glu 35 4045 Val Lys Ala Ser Gly Asn Thr Phe Gly Asn Tyr Phe Gln Val Ala Thr 50 5560 Tyr Gly Glu Ser His Gly Gly Gly Val Gly Cys Val Ile Ser Gly Cys 65 7075 80 Pro Pro Arg Ile Pro Leu Thr Glu Ala Asp Leu Gln Val Glu Leu Asp 8590 95 Arg Arg Arg Pro Gly Gln Ser Arg Ile Thr Ser Thr Arg Lys Glu Thr100 105 110 Asp Thr Cys Lys Ile Leu Ser Gly Thr His Glu Gly Val Thr ThrGly 115 120 125 Thr Pro Ile Leu Val Ile Val Pro Asn Thr Asp Gln Ile GlySer Asp 130 135 140 His Arg Glu Ile Ala Asn Val Tyr Arg Pro Ser His AlaAsp Ala Thr 145 150 155 160 Tyr Asp Phe Lys Tyr Gly Val Arg Ala Val GlnGly Gly Gly Arg Ser 165 170 175 Ser Gly Arg Lys Thr Val Gly Arg Val AlaAla Gly Ala Leu Pro Lys 180 185 190 Lys Ile Leu Lys Leu Lys Cys Gly LeuGlu Ile Leu Ser Phe Val Ser 195 200 205 Lys Val His Gln Val Val Leu ProGlu Asp Ala Val Asp Tyr Gly Ser 210 215 220 Val Thr Leu Glu Gln Ile GluSer Asn Ile Val Arg Cys Pro Asp Pro 225 230 235 240 Glu Tyr Ala Glu LysMet Ile Asp Ala Ile Asp Arg Val Arg Val Arg 245 250 255 Gly Asp Ser ValGly Gly Val Ile Thr Cys Val Ala Arg Asn Val Pro 260 265 270 Arg Gly LeuGly Ser Pro Val Phe Asp Lys Leu Glu Ser Glu Leu Ala 275 280 285 Lys AlaMet Leu Ser Ile Pro Ala Ser Asn Gly Phe Glu Ile Gly Ser 290 295 300 GlyPhe Ala Gly Thr Asp Leu Thr Gly Ser Glu His Asn Asp Glu Phe 305 310 315320 Tyr Met Asp Lys Ala Gly Ser Val Arg Thr Arg Thr Asn Arg Ser Gly 325330 335 Gly Val Gln Gly Gly Ile Ser Asn Val Glu Ile Val His Phe Lys Val340 345 350 Ala Phe Lys Pro Thr Pro Ser Ile Gly Val Lys Gln Asn Thr ValSer 355 360 365 Arg Glu Arg Gln Asn Val Glu Leu Leu Ala Arg Gly Arg HisAsp Pro 370 375 380 Cys Val Ala Pro Arg Ala Val Pro Val Val Glu Ser MetAla Ala Leu 385 390 395 400 Val Leu Met Asp Gln Leu Met Ala His Val AlaGln Cys Glu Met Phe 405 410 415 Ala Leu Asn Thr Ala Leu Gln Glu Pro ValGly Ser Phe 420 425 9 479 DNA Oryza sativa unsure (243)..(244) n = A, C,G, or T 9 tgtatcaaga aacaacatac tgtttcaagg gagcatgagg atgttgaacttttagcaagg 60 ggccgccacg acccatgtgt tgtccctcgc gctgtgccga tggtggagtccatggccgca 120 ttagtcctca tggaccagct gatggcgcac attgctcaat gtgagatgtttccactgaac 180 cttgctctac aagaaccagt tggctctgcc agcagcgtac ctgcatttgcaccagatcta 240 aannggnccc ccctcccccc cccccagctt gtttatcatc tatcatatttctggggtttt 300 ctaaggggtt cgcagttttg ccacaaagcc tgtatcctag tttatatctcgagttattgt 360 acccaaggaa tccgttatac agtgagcatg aagatagaaa tgcgttcatgcgtgttttgt 420 gatatggaca atctgtgctt acatcaagtt attttgagca ataaaaatcncaatttatg 479 10 81 PRT Oryza sativa 10 Cys Ile Lys Lys Gln His Thr ValSer Arg Glu His Glu Asp Val Glu 1 5 10 15 Leu Leu Ala Arg Gly Arg HisAsp Pro Cys Val Val Pro Arg Ala Val 20 25 30 Pro Met Val Glu Ser Met AlaAla Leu Val Leu Met Asp Gln Leu Met 35 40 45 Ala His Ile Ala Gln Cys GluMet Phe Pro Leu Asn Leu Ala Leu Gln 50 55 60 Glu Pro Val Gly Ser Ala SerSer Val Pro Ala Phe Ala Pro Asp Leu 65 70 75 80 Ser 11 966 DNA Oryzasativa 11 cacgagtaca gcctctcacc aaccaaacca acaacctcgc tccgatggccgcgccaacgt 60 cgtcgcagcc ggtggcgcgc gtcctccccc gcggcggcgg cggcgggttccgcgccttcc 120 cggagtccgc cccggcttcc ctccgcttct ccgtcggccg ccgccgcgccgctcgcctag 180 aggtgaaggc gtctgcaaat gtatttggga actacttcca ggttgcaacttatggagagt 240 ctcatggagg cggtgttggt tgcgtaatca gtggatgccc acccagaatcccacttactg 300 aagcagatat gcaagtagaa ctcgaccgga gacggccagg ccagagcagaataaccaccc 360 caagaaagga gactgacact tgcaaaattc tttcagggac acatgaaggaatgaccactg 420 ggacaccaat tcatgttttt gtcccgaaca cagatcagag agggggtgattacagtgaaa 480 tggctaaggc ctacagacct tcacatgcag atgcaactta tgacttcaaatacggtgtta 540 gagcagtgca gggaggtgga agatcatcag caagagagac cattggaagggtggctgcag 600 gagctcttgc aaagaaaatt cttaagctca aatctggagt agagatcttggcgtttgtgt 660 ccaaggtgca tcaagttgta ctaccagaag atgccgttga ttatgacactgtaacaatgg 720 aacagataga aagcaacatt gttagatgtc ctgatccaga atatgcacagaagatgattg 780 atgcactcga taaagtacga gttagaggtg attcgattgg tggtgtggtcacatgcattg 840 caagaaatgt tcctcgtggg attggctctc ctgtatttga caaacttgaggctgaattgg 900 cgaaagctat gctttctctt cctgcaagca aggggtttga gatcggcagtggatttgtgt 960 tcacta 966 12 307 PRT Oryza sativa 12 Met Ala Ala Pro ThrSer Ser Gln Pro Val Ala Arg Val Leu Pro Arg 1 5 10 15 Gly Gly Gly GlyGly Phe Arg Ala Phe Pro Glu Ser Ala Pro Ala Ser 20 25 30 Leu Arg Phe SerVal Gly Arg Arg Arg Ala Ala Arg Leu Glu Val Lys 35 40 45 Ala Ser Ala AsnVal Phe Gly Asn Tyr Phe Gln Val Ala Thr Tyr Gly 50 55 60 Glu Ser His GlyGly Gly Val Gly Cys Val Ile Ser Gly Cys Pro Pro 65 70 75 80 Arg Ile ProLeu Thr Glu Ala Asp Met Gln Val Glu Leu Asp Arg Arg 85 90 95 Arg Pro GlyGln Ser Arg Ile Thr Thr Pro Arg Lys Glu Thr Asp Thr 100 105 110 Cys LysIle Leu Ser Gly Thr His Glu Gly Met Thr Thr Gly Thr Pro 115 120 125 IleHis Val Phe Val Pro Asn Thr Asp Gln Arg Gly Gly Asp Tyr Ser 130 135 140Glu Met Ala Lys Ala Tyr Arg Pro Ser His Ala Asp Ala Thr Tyr Asp 145 150155 160 Phe Lys Tyr Gly Val Arg Ala Val Gln Gly Gly Gly Arg Ser Ser Ala165 170 175 Arg Glu Thr Ile Gly Arg Val Ala Ala Gly Ala Leu Ala Lys LysIle 180 185 190 Leu Lys Leu Lys Ser Gly Val Glu Ile Leu Ala Phe Val SerLys Val 195 200 205 His Gln Val Val Leu Pro Glu Asp Ala Val Asp Tyr AspThr Val Thr 210 215 220 Met Glu Gln Ile Glu Ser Asn Ile Val Arg Cys ProAsp Pro Glu Tyr 225 230 235 240 Ala Gln Lys Met Ile Asp Ala Leu Asp LysVal Arg Val Arg Gly Asp 245 250 255 Ser Ile Gly Gly Val Val Thr Cys IleAla Arg Asn Val Pro Arg Gly 260 265 270 Ile Gly Ser Pro Val Phe Asp LysLeu Glu Ala Glu Leu Ala Lys Ala 275 280 285 Met Leu Ser Leu Pro Ala SerLys Gly Phe Glu Ile Gly Ser Gly Phe 290 295 300 Val Phe Thr 305 13 541DNA Glycine max unsure (411) n = A, C, G, or T 13 ctcaatcaat ctaattctcccatttctctt ccaatggcgt cttctctttc caccaaacca 60 ttctcagccg acgctctctccgccttcgct tctctcaatt ccgatctcgg atccctctcc 120 cccgcctacc tccgactctcactccgtcct cgtcttccca agagacttca catacaggcg 180 gctgggagta cctatggaaatcactttcgt gttacaacat atggggaatc acatggagga 240 ggtgttggtt gtgttattgatggatgtcct cctcgccttc ctctctctga agctgatatg 300 caagtggatc ttgacagaaggaggccaggt cagagccgaa ttacaactcc tagaaaggag 360 actgatacat gtaaaatattttcaggagtt tccgaaggaa tcactactgg nactccaatt 420 catgtactgt acccanntactgatcaanga gggcatgact atagcnagat ggnagtacnt 480 ataggccccc catgcaatgnaccntgacat gaactatggg tngatagtta aggtggnggg 540 g 541 14 168 PRT Glycinemax UNSURE (139) Xaa = ANY AMINO ACID 14 Met Ala Ser Ser Leu Ser Thr LysPro Phe Ser Ala Asp Ala Leu Ser 1 5 10 15 Ala Phe Ala Ser Leu Asn SerAsp Leu Gly Ser Leu Ser Pro Ala Tyr 20 25 30 Leu Arg Leu Ser Leu Arg ProArg Leu Pro Lys Arg Leu His Ile Gln 35 40 45 Ala Ala Gly Ser Thr Tyr GlyAsn His Phe Arg Val Thr Thr Tyr Gly 50 55 60 Glu Ser His Gly Gly Gly ValGly Cys Val Ile Asp Gly Cys Pro Pro 65 70 75 80 Arg Leu Pro Leu Ser GluAla Asp Met Gln Val Asp Leu Asp Arg Arg 85 90 95 Arg Pro Gly Gln Ser ArgIle Thr Thr Pro Arg Lys Glu Thr Asp Thr 100 105 110 Cys Lys Ile Phe SerGly Val Ser Glu Gly Ile Thr Thr Gly Thr Pro 115 120 125 Ile His Val SerVal Pro Asn Thr Asp Gln Xaa Arg His Asp Tyr Ser 130 135 140 Glu Met AlaLeu Leu Ile Gly Leu His Ala Asn Ala Thr Tyr Asp Met 145 150 155 160 LysTyr Gly Xaa Arg Ser Val Lys 165

What is claimed is:
 1. An isolated nucleic acid fragment encoding achorismate synthase comprising a member selected from the groupconsisting of: (a) an isolated nucleic acid fragment comprising at least500 nucleotides wherein the nucleic acid fragment hybridizes to anisolated nucleic acid fragment encoding the amino acid sequence setforth in a member selected from the group consisting of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ IDNO:14; (b) an isolated nucleic acid fragment that is complementary to(a).
 2. The isolated nucleic acid fragment of claim 1 wherein nucleicacid fragment is a functional RNA.
 3. The isolated nucleic acid fragmentof claim 1 wherein the nucleotide sequence of the fragment comprises thesequence set forth in a member selected from the group consisting of SEQID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11 and SEQ ID NO:13.
 4. A chimeric gene comprising the nucleic acidfragment of claim 1 operably linked to suitable regulatory sequences. 5.A transformed host cell comprising the chimeric gene of claim
 4. 6. Achorismate synthase polypeptide comprising all or a substantial portionof the amino acid sequence set forth in a member selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12 and SEQ ID NO:14.
 7. A method of altering the levelof expression of a chorismate synthase in a host cell comprising: (a)transforming a host cell with the chimeric gene of claim 4; and (b)growing the transformed host cell produced in step (a) under conditionsthat are suitable for expression of the chimeric gene wherein expressionof the chimeric gene results in production of altered levels of achorismate synthase in the transformed host cell.
 8. A method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding a chorismate synthase comprising:(a) probing a cDNA or genomic library with the nucleic acid fragment ofclaim 1; (b) identifying a DNA clone that hybridizes with the nucleicacid fragment of claim 1; (c) isolating the DNA clone identified in step(b); and (d) sequencing the cDNA or genomic fragment that comprises theclone isolated in step (c) wherein the sequenced nucleic acid fragmentencodes all or a substantial portion of the amino acid sequence encodinga chorismate synthase.
 9. A method of obtaining a nucleic acid fragmentencoding a substantial portion of an amino acid sequence encoding achorismate synthase comprising: (a) synthesizing an oligonucleotideprimer corresponding to a portion of the sequence set forth in any ofSEQ ID NOs:1, 3, 5, 7, 9, 11 or 13; and (b) amplifying a cDNA insertpresent in a cloning vector using the oligonucleotide primer of step (a)and a primer representing sequences of the cloning vector wherein theamplified nucleic acid fragment encodes a substantial portion of anamino acid sequence encoding a chorismate synthase.
 10. The product ofthe method of claim
 8. 11. The product of the method of claim
 9. 12. Amethod for evaluating at least one compound for its ability to inhibitthe activity of a chorismate synthase, the method comprising the stepsof: (a) transforming a host cell with a chimeric gene comprising anucleic acid fragment encoding a chorismate synthase, operably linked tosuitable regulatory sequences; (b) growing the transformed host cellunder conditions that are suitable for expression of the chimeric genewherein expression of the chimeric gene results in production of thechorismate synthase encoded by the operably linked nucleic acid fragmentin the transformed host cell; (c) optionally purifying the chorismatesynthase expressed by the transformed host cell; (d) treating thechorismate synthase with a compound to be tested; and (e) comparing theactivity of the chorismate synthase that has been treated with a testcompound to the activity of an untreated chorismate synthase, therebyselecting compounds with potential for inhibitory activity.